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Bacterial BRIDGEs

Communication capability is a key component of the modern information-driven world. From internet to instant messenger, SMS to telephone, society has developed a large number of technological means for people to keep in constant contact with one another and to exchange information ranging from the trivial to the complex. Even simple speech, and communication of ideas via concepts and words, is a key differentiating factor between human beings and other higher mammals.

What if bacteria such as E. coli were able to communicate via a means more efficient than simple chemical signalling? The creation and sensing of light is not a novel idea in synthetic biology, as evidenced by the firefly luciferase reporter developed by Ljubljana 2007 and the photoreceptor submitted by Lausanne 2009. Until now, however, there has not been a concentrated effort to match light production with light reception. The 2010 University of Edinburgh iGEM team has worked to create a standardised set of light producing and light sensing BioBricks with which light-based communication can take place.

FORTH stands for Fabricated Organism Reception and Transmission of Heterogeneous light. It establishes a core set of BioBricks that allow synthetic organisms to create light of a determined wavelength upon a specified stimulus, and to activate a specified response when they sense said light.

Our Project

Figure 1: Normalised absorbance spectra of:
a. the Lov2 domain of Avena sativa with bound FMN. Adapted from Schüttrigkeit et al. (2003).
b. Green light absorbing form of cyanobacteriochrome CcaS from Synechocystis sp. PCC 6803. Adapted from Hirose et al. (2008).
c. Red light absorbing form of phytochrome Cph1-PCB adduct, from Synechocystis sp. PCC 6803. Adapted from Gambetta and Lagarias (2001).
Note that the relative absorbance of each spectrum is normalised to one.

Figure 2: Normalised emission spectra of:
a. bacterial luciferase LuxAB from V. campbellii. Adapted from Suadee et al. (2003).
b. firefly luciferase from P. pyralis, wildtype. Adapted from Shapiro et al. (2009).
c. firefly luciferase from P. pyralis, substitution mutant S284T. Adapted from Branchini et al. (2007).
Note that the relative emission of each spectrum is normalised to one.

The response of light receptors Cph8, LovTAP and our green light receptor to different wavelengths of light have not been fully characterised. The absorbance spectra of the light sensitive proteins (Figure 1) might not exactly mirror their response to light, but should give us a good idea of their properties until this is possible.

In LovTAP, the light sensitive domain of the protein is the Lov2 domain from the Avena sativa blue light receptor phototropin. This binds flavin mononucleotide (FMN) which is its co-factor. Schüttrigkeit et al. (2003) measured the absorbance of the wildtype Lov2 bound to FMN. Since this is the active part of LovTAP, we expect our blue light receptor to have a similar response in vivo. The red light absorbing form of Cph1 is what responds to red light in Cph8. The absorbance of Cph1 was measured by Gambetta and Lagarias (2001). Similarly, the absorbance of the green light absorbing form of CcaS, which we are planning on using in our green light receptor, was measured by Hirose et al. (2008).

In accordance with the above, the challenge was then to design and characterise luciferases compatible with the sensors. In this, we decided to build on previous work by Ljubljana 2007 and Edinburgh 2009. The firefly luciferase from Photinus pyralis deposited as a BioBrick by the former formed the base of our red and green light producing proteins, while the bacterial luciferase LuxAB from Xenorhabdus luminescens BioBricked by last year's Edinburgh team was our blue light protein.

The emission peak of the wildtype firefly luciferase is roughly 557nm at pH 7.8 according to Branchini et al. (2005). We needed to mutate this towards the red spectrum in order to activate the Cph8-based red light sensor, and also proposed to introduce a mutation towards the green spectrum so as to better match it with our hypothetical green light sensor. The LuxAB-LumP fusion already available in the Registry was theoretically already of the correct wavelength to activate the blue light sensor, although further characterisation was necessary. The theoretical spectra of the luciferases we sought to create are displayed in Figure 2.

Difficulties were foreseen in making the various light producing proteins bright enough to activate their corresponding sensors, as well as in providing the substrates necessary for the bacteria to constantly emit light. However, once we received word that this year's Cambridge team was working to alleviate these problems, we decided to focus our efforts on the process of creating and characterising the various light producing and light sensing proteins.

During the course of the project, we collaborated closely with the UNAM-Genomics team from Mexico, since it was apparent from an early stage that we were pursuing very closely related ideas.

Table of Contents

Throughout this wiki there are words in bold that indicate a relevance to human aspects. It will become obvious that human aspects are a part of almost everything in iGEM.